Methods for isoelectric particle separation

ABSTRACT

In order to isoelectrically separate particles with a pH-dependent net charge, the particles are exposed in a guiding liquid to electric field forces. The pH value of the guiding liquid is set in such a way that at least one predetermined type of particle is separated from the remaining particles and migrates to a fixing collecting means under the effect of the electric field forces. The collecting means is for example a porous hollow fiber delimited by electrodes which generate the electric field forces and crossed by the guiding liquid together with the sample to be separated. The particles whose isoelectric point matches the pH value of the guiding liquid run unimpeded through the fibers, whereas the remaining particles are pressed against the inner wall of the fiber and are prevented from being carried away with the liquid flow.

This application is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/EP97/05295 which has an Internationalfiling date of Sep. 26, 1997 which designated the United States ofAmerica.

The invention concerns methods and devices for isoelectric separation ofparticles whose charge characteristics depend on the pH value of aguiding fluid, especially for separating ampholytic suspended particles,colloids or biological cells. The invention concerns in particular theseparation of such particles from a guiding fluid flow.

Numerous separation techniques are known from molecular biology;biochemistry, medicine and biotechnology whose function is based oncharge differences of molecules within a substance that is to beseparated. In the case of amphoteric ion compounds (socalled ampholytesor ampholytic molecules), the molecular charge depends on the pH valueof the surrounding solution. The isoelectric point (hereafter referredto as the IP) of a compound is the pH value at which the net charge ofthe amphoteric molecules equals zero. The charge of the molecule ispositive for a pH value smaller than the IP and negative in the oppositecase.

In isoelectric focusing (hereafter referred to as IEF), proteins withdifferent IPs are separated by making use of spatial pH gradients alonga separation length (see R. A. M. Osher et al. in “The Dynamics ofElectrophoresis”, published by B. J. Radola, VCH, Weinheim, 1992, pp163-231). IEF is performed, for example, using porous gel matrices or,for analytical separation of the smallest samples, using thincapillaries as what is called cIEF (see F. Foret et al. “CapillaryElectrophoresis” in “Advances in Electrophoresis”, vol. III, publishedby A. Chrambach et al., VCH, Weinheim, pp 273-347).

For the following reasons conventional IEF presents disadvantages andits use is restricted. Retrieval of the separated substances from acarrier material, especially for further processing like analysis,medication applications or the like, calls for elaborate procedures bywhich the separated substances themselves may be modified, or which leadto substance losses. The necessary use of additional substances to forma pH gradient that is as wide and linear as possible producesrestrictions in terms of further use of the separated samples. Thesocalled carrier ampholytes used as additional substances are, inchemical terms, a highly diverse substance mix that is difficult toseparate from the separated protein fractions. Furthermore, cIEF isrestricted to minimal sample quantities that cannot be collectedseparately and are not separable from the carrier ampholytes.

The problem when socalled IPG membranes are used to form the pH gradientis that proteins, because of the necessity of passing through such amembrane, must pass through a milieu whose ionic concentration is verylow. Consequently, sensitive proteins can denaturize or precipitate inthis region. For this reason the strictest requirements are made in IEFfor uniformity of the voltage gradients in the separation length.Finally, problems can occur in IEF in the form of electroosmosis inextreme pH ranges, the carrier medium (gel) heating up and altering(destruction of the gel) for instance.

To overcome such drawbacks, systems were developed in which ampholyteseparation is produced by the effect of an external electric field andwithout modifying additional substances. One separating system is known,for example, that works by the method of socalled electric field flowfractionating (eFFF) (see K. D. Caldwell et al. in “Science”, vol. 176,1972, pp 296-298). In this separating system a continuous fluid streamis conducted through a narrow duct between two ion flow permeablemembranes, into which the sample to be separated is injected as a narrowband. External electrodes, in electrical contact with the duct through asurrounding fluid, produce different degrees of retardation, as afunction of charge, of the protein molecules in the fluid stream.However, use of this method is restricted to protein molecules with IPsthat are relatively far apart. Furthermore, no complete proteinseparation could be achieved. Control of the pH value of the fluidstream—and thus of the molecular charge state—is not a facet of thisfamiliar separating system.

The eFFF system is not applicable in practice. Although the samplescould be separated (incompletely), collection of the separated fractionswas not implemented. Furthermore, the times for separation areunacceptably high. A follow-on development of the above mentioned eFFFsystem (see L. F. Kesner et al. in “Analytical Chemistry”, vol. 48,1976, pp 1834-1839) produced analysis or separation times of severalhours even on a laboratory scale for example.

A modified eFFF system described by Lightfoot et al. (see “SeparationScience and Technology”, vol. 16, 1981, pp 619-656) makes use offcylindrical duct geometry. The separation performance of this system wasalso unacceptable in practical terms. Furthermore, protein retardationshowed itself to be a complex function of a large number of parameters,eg the buffer ions that were used, the sample quantity, the sort ofprotein and properties of the duct wall. Nor is this familiar systemintended for pH control or, say, separation of proteins with differentIPs.

There is pronounced interest in substance separation for the productionof high-purity substances, beyond the sphere of the laboratory, on anindustrial scale. Because of the restrictions and disadvantagesmentioned however, no continuous substance separation is known to date,using the named systems, that produces adequate separation performanceand speed for the practical sphere.

The object of the invention is to indicate improved methods ofisoelectric particle separation distinguished, in particular, by higherseparation speed, greater reliability, and a broader range ofapplication in terms of separable particles and the surroundingsolutions. The object of the invention is also to indicatecorresponding, continuous isoelectric purification methods. The objectof the invention also consists in providing devices for implementing themethods of particle separation and further application possibilities.

The named objects are achieved by methods for isoelectric particleseparation in which particles with a net charge or charge density thatis a function of the pH value of the surroundings are exposed toelectric field forces in a guiding fluid passing by collection means,whereby the pH value of the guiding fluid is set so that, through theeffect of the electric field forces, at least one predetermined particletype undergoes a change in motion as a function of charge and is movedto the collection means, intended for soluble fixing of the chargedparticles or by devices for isolectric particle separation that include:electrode means for forming an electric field in a guiding fluid withparticles whose net electric charge or charge density is a function ofthe pH value of the guiding fluid; pH setting means being adapted to setthe pH value of the guiding fluid so that at least one predeterminedparticle type of the particles in the guiding fluid undergoes a changein motion as a function of charge through the effect of the electricfield; and collection means arranged between the electrode means and theguiding fluid for soluble fixing of charged particles. Preferredembodiments of the invention are disclosed hereinafter.

The separation technique according to the invention is based on the ideaof controlling or setting the pH value in a guiding fluid in which theparticles to be separated are exposed to electric field forces so thatall particle types with a net charge go through a motion dependent oncharge, and the remaining, for the most part neutral particles show nochange in their state of motion, whereby the particles moving as afunction of charge are moved to collection means (collection device).The particles are retained at least temporarily on the collection means.This involves fixing on the surface or in the volume of the collectionmeans. The duration of fixing is determined by the separationconditions, especially modulation with time of the electric fieldforces, alteration of the pH value, the structure of the collectionmeans and/or relative motion between the collection means and theguiding fluid.

A major difference from the eFFF technique described above is thus thatthe particles are not differently retarded as a function of charge butthat, through control of the pH value in the guiding fluid, a conditionis created that defines what type of particle, exposed to the effect ofan external electric field, moves into an at least temporarily fixedstate and is possibly released again. To distinguish it from the eFFFtechnique, isoelectric separation according to the invention istherefore named pH-controlled electroretention chromatography. Thecollection means are effective as real restrain means. Thus a method isproposed for inverse isoelectric particle separation in which particleswith a charge dependent on pH value are passed by collection means in aguiding fluid and the pH value of the guiding fluid is set or modifiedso that only the uncharged particles remain in the guiding fluid, whilethe charged particles are moved through an electric field to thecollection means and are consequently fixed, at least temporarily, as afunction of pH value.

The induced change of motion of the predetermined type of particle,according to the invention, is directed to the collection means so thatthe predetermined type of particle that is to be separated arrangesitself or collects on or behind the collection means. The collectionmeans are formed of a collection arrangement, for example, betweenelectrode means to generate the external electric field and the guidingfluid. It is possible to move the collection means in relation to theguiding fluid.

The collection arrangement is preferably semi-permeable as a function ofsubstance. Semi-permeability can mean, for instance, that the collectionarrangement is permeable for the molecules of the guiding fluid or forions dissolved in the guiding fluid, but impermeable, ie a barriereffect, for the substance or type of particle to be separated.Semi-permeability can also be implemented so that some of the type ofparticle to be separated with smaller particle sizes are let through,and the remainder of the particles to be separated with larger particlesizes are not let through. The collection arrangement is preferably adelimitation of the guiding fluid from the particles to be separated.Outside of the collection arrangement, in a surrounding solution withadjustable pH value, are the electrode means for forming the electricfield.

The invention can be implemented with a static or a flowing guidingfluid. In both cases the collection arrangement forms a compartment or avessel with at least one opening for entry or exit of the sample to beseparated.

The separating effect is especially good and fast if the displacementpath of the particles from the location of the sample in the guidingfluid (possibly flowing tangentially to the adjoining collection means)to the collection means is kept as small as possible. Preferably thecollection arrangement will have characteristic dimensions that are inthe flow direction of the guiding fluid much greater than thedisplacement path. The displacement path or inner dimensions of thecompartments are of the order of mm or smaller for example.

In one preferred embodiment, the collection arrangement is formed of atleast one longish, hollow element (eg tubular) that has semi-permeableor porous walls and through which the guiding fluid flows with theparticle sample to be separated. Other forms of collection arrangementare also possible, especially with a rectangular instead of a roundcross-section. In one particularly advantageous and preferredimplementation the collection arrangement consists of at least onestraight or bent hollow fiber with, at least in part, semi-permeablewalls.

Isoelectric separation according to the invention can be implementedwith ampholytic molecules or all other synthetic or biological particles(especially cells or viruses) that exhibit electrical characteristicslike those of ampholytic molecules, in particular a net charge or chargedensity that is a pH function of the surroundings.

Optionally, in order to purify the guiding fluid, particles that are tobe removed from the guiding fluid may be moved out of the guiding fluidby the collection means. The flow velocity of the guiding fluid may bereduced or dropped to zero at least temporarily when the particles to beseparated are exposed to the electric field forces. A sample with theparticles to be separated and/or the guiding fluid may be fed throughmicroducts in the porous wall. The pH value of a sample solution, withwhich the particles are introduced to the guiding fluid, of the guidingfluid and/or of the surrounding solution may be set independently sothat predetermined pH gradients form on the collection means. The pHvalue of a sample solution, in which the particles to be separated areintroduced to the guiding fluid, and/or of the guiding fluid may be setby diffusive exchange processes with the surrounding solution.

Depending on the application, the purpose of isoelectric separationaccording to the invention can be aimed at obtaining the predeterminedparticle type that undergoes a change of motion in the electric field asa function of charge, or the remaining particles without change of thestate of motion.

A device for isoelectric particle separation according to the inventioncontains electrode means for forming an electric field in a guidingfluid with the particles that are to be separated, and means forcontrolling or setting the pH value in the guiding fluid. There are alsocollection means between the electrode means and the guiding fluid that,in addition to a collecting function for a type of particle that is tobe separated, also have a delimiting function from a surroundingsolution with adjustable pH value. The collection means are permeable atleast for ions dissolved in the surrounding solution and the guidingfluid, so that the pH value of the guiding fluid can also be set andcontrolled by setting the pH value of the surrounding solution.

Preferred forms of application of the invention are all separationprocesses on sample mixes that contain at least one particle type withampholytic characteristics, and/or investigations of certain substancesfor determining the isoelectric point, of titration curves oraggregation response. The device according to the invention can also beused to reduce the ionic concentration of the guiding fluid in the fieldof pH-controlled electrodialysis.

The present invention provides also a device for isolectric particleseparation that includes: electrode means for forming an electric fieldin a guiding fluid with particles whose net electric charge or chargedensity is a function of the pH value of the guiding fluid; pH settingmeans being adapted to set the pH value of the guiding fluid so that atleast one predetermined particle type of the particles in the guidingfluid undergoes a change in motion as a function of charge through theeffect of the electric field; and collection means arranged between theelectrode means and the guiding fluid for soluble fixing of chargedparticles.

Isoelectric particle separation according to the invention possesses thefollowing advantages:

Fractionating or separation occurs without additional substances, inparticular without carrier ampholytes or other means such as forgenerating a pH gradient for instance. Since no pH gradient isgenerated, this simplifies considerably a preparative procedure in theflow-through system or collection of the required (possibly even all)separated fractions. The separated or purified fractions are availablefor further manipulation after separation, without having changed fromthe original form. The separated fractions are, in particular, always inthe same solution with the same, possibly slightly reduced or evenhigher concentration. The system according to the invention can be usedand automated for both discontinuous and continuous separation ofparticles as a function of their IPs. Both spatial fractionation and, byvariation of the pH value during the separation process, fractionationas a function of time is possible. The speed of separation is high, ofthe order of minutes, as will be exemplified in what follows. Theseparation system allows configuration of the electrode means withintight confines so that, compared to conventional separation processes,sufficiently high field strength can be produced with relatively smallvoltages. This minimizes the energy consumption of systems according tothe invention and relaxes conditions for samples sensitive totemperature.

Another aspect of the present invention is the use of a device forseparation of at least one particle type from the guiding fluid toobtain a purified fraction of the separated particles and/or a purifiedguiding fluid, determining electrical and thermodynamic characteristicsof substances like isoelectric point, titration curves or aggregationresponse, boosting the concentration of a particle solution orsuspension, or reducing the ionic concentration of the guiding fluid.

Further features and advantages of the invention will emerge from thefollowing description of implementations referring to the attacheddrawings.

FIG. 1: Schematic explaining the forces that appear in separationaccording to the invention,

FIG. 2: Curve illustrating protein separation according to theinvention,

FIG. 3: Curve illustrating further protein separation according to theinvention,

FIG. 4: Curve of a model calculation illustrating the selectivity thatcan be achieved with the invention,

FIG. 5: Curve illustrating further protein separation according to theinvention,

FIG. 6: Schematic of a separation device according to the invention,

FIGS. 7, 8, 9: Schematics of modified hollow fiber arrangements,

FIG. 10: Schematics of combined separating and concentrating device,

FIG. 11: Schematic of multi-compartment system according to theinvention, and

FIG. 12: Schematic of modified implementation of a separation deviceaccording to the invention.

The following explanation refers, by way of example, to animplementation of the invention in which the collection means are formedof a hollow fiber. But the principles explained can be implemented inthe same way in other geometries of the collection means. Furthermore,the invention is not restricted to protein separation as used here as anexample, it can be implemented for any particles with ampholyticcharacteristics.

In one implementation of the invention (see below for details, FIG. 6),a thin hollow fiber passes through a reaction cavity containing thesurrounding solution, at least two electrodes and means for pH setting.The electrodes are designed for application of a pedetermined DCvoltage. The walls of the hollow fiber are provided with a large numberof pores of defined size to create semi-permeability. The size of thepores is chosen so that the solution (eg water) and ions transportingthe electric current are let through, but the molecules to be separatedare usable to pass through the wall. The hollow fiber can for example,have an inner diameter (or clear width or socalled lumen) in the rangeof millimeters to micrometers. The means for pH setting are provided tocontrol or set the pH value of the surrounding solution by what is afamiliar, conventional procedures eg electrically or by titration.

The sample to be separated is conducted through the lumen of the hollowfiber with a second fluid, the guiding fluid. The guiding fluid willpreferably have the same pH value or the same ion composition as thesurrounding solution. For this purpose the guiding fluid can beextracted from the reaction cavity for example. Alternatively it ispossible for the guiding fluid to have a different pH value. At theupstream end of the hollow fiber there are means for introducing thesample to be separated into the guiding fluid, and at the downstream endthere are means of detection and/or collection. The forces appearingduring separation according to the invention will now be explained withreference to FIG. 1.

FIG. 1 is a schematic section of a portion of a hollow fiber 11 throughwhich the guiding fluid with a certain pH value flows and the sample 13a, 13 b and 13 c that is to be separated. The hollow fiber 11 isarranged between two DC electrodes 12 a, 12 b so that the guiding fluidwith the sample passes through an electric field.

According to the pH value of the guiding fluid, the sample comprisesnegatively charged molecules 13 a, positively charged molecules 13 b anduncharged molecules 13. Exposed to the effect of the electric fieldforces, the charged molecules move to the edge of the guiding fluid. Atthe edge of the fluid flow, ie on the hollow fiber acting as thecollection means, the charged molecules are prevented from beingtransported further with the guiding fluid and thus locally fixed. Localfixing can generally be achieved by a sufficiently strong electric fieldthat has one component perpendicular to the direction of flow of theguiding fluid (V_(h)) and by an adhering (eg rough or porous) innersurface of the hollow fiber. In a preferred implementation of theinvention the geometry of the hollow fiber and the flow velocity of theguiding fluid are selected so that the flow profile illustrated in FIG.1 assumes what an essentially parabolic shape. Such a flow profile ischaracterized by the fact that flow velocity is highest in the middle ofthe guiding fluid, ie in the center of the hollow fiber, reducingtowards the edges. Such a flow profile is produced, for example, whengenerating a laminar guiding fluid flow through the hollow fiber. Flowvelocity is zero in the immediate vicinity of the hollow fiber wall. Inthis case the molecules are excluded entirely from further transport inthe guiding fluid flow (electroretention).

A molecule whose IP matches the pH value of the guiding fluid possessesno net electric charge and consequently is unaffected by the electricfield. Thus, as a function of the pH value, a certain type of moleculeis conducted through the hollow fiber to a detector or collector, forexample, while the charged particles on the hollow fiber wall areretained.

FIG. 2 and FIG. 3 show experimental results for protein separation. Thecurves are absorption values recorded by a detector as a function oftime at the downstream end of the hollow fiber. At point 1 in FIG. 2 adefined amount of protein is introduced to the guiding fluid anddetected without the effect of an electric field as a function of time(max. 5 min). At point 2 an amount of protein is again introduced to theguiding fluid, but this time with the effect of an electric field. Thereis no change in absorption—or at least only a slight negative oneproduced by the measuring system—because the protein is retained. Theretained protein does not enter the carrier flow again, and is thusdetected (max. 20 min), until the electric field is removed.

FIG. 3 shows an experiment where the protein has an IP that correspondsto the pH value of the surrounding solution or guiding fluid however.Between points 1 and 2 (without the influence of a field) and 2 and 3(with the influence of a field) it can now be seen that the proteinremains unaffected because of the absence of a net charge and reachesthe detector unaltered. When the field is removed (point 3), noabsorption of the protein is detected but instead only the impuritiescontained in the starting sample. This shows the purifying effectproduced by passing the protein through the electric field, in thecourse of which the impurities were withdrawn from the sample.

In addition to the separation of proteins with an IP that differs fromthe pH value according to FIG. 2, or the separation of impuritiesaccording to FIG. 3, the following variants are possible of the methodsaccording to the invention.

It is possible to alter the pH value of the surrounding solution andthus of the guiding fluid with time. If variation with time is attunedto the length of the hollow fiber or the flow velocity of the guidingfluid, it is possible, under the continuous influence of a field and byvarying the pH value, to separate components from the sample with a timeoffset, ie in the order of their IPs. Controlled, carrier-free pHvariation can be produced over a wide range with accuracy of approx. 0.1to 0.01 pH units with one pH state. In this timed fractionating the pHvalue is thus increased or reduced as a function of time in such a waythat the pH value corresponds to the IP of a component of the sample fora certain time interval. The time interval is selected so that therewill be complete separation of the particular component from the sampleand spatial separation on the inner wall of the hollow fiber from thenext component (fractionating of a substance mix).

In addition to pH variation for charge variation of the molecules orparticles, it is possible to provide, instead of the above mentioned DCvoltage between the electrodes, modulation of the electric field in timeand/or space. The result is again an optimization in separation of thesample.

Another alternative is variation of the hydrodynamic flow through thehollow fiber (variation of flow velocity). In this way it is possible torestrict the flow of the guiding fluid with time or to halt it while thesample is exposed to the effect of the field in the hollow fiberelement.

One variation of a separating or purifying process according to theinvention is possible when a semi-permeability of the hollow fiber(choice of pore size) is created so that at least part of the releasedmolecules that are to be separated are allowed to pass. A pH value isset in the surrounding solution of the guiding fluid that corresponds tothe IP of the component to be isolated. After injection of the sample asa narrow band or in continuous application, all components whose IP doesnot correspond to the set pH value are shifted out of the hollow fiberlumen through the pores of the wall into the surrounding solution. Thecomponent, now purified, remaining the hollow fiber can be collectedwith the guiding fluid stream after the isolating gap. The concentrationof the purified component only decreases slightly because of diffusionthrough the wall of the hollow fiber, but it can be increased again by afollow-up concentration booster (see FIG. 10).

FIG. 4 shows the result of a model calculation to illustrate the effectof the electric field on molecules whose IP does not correspond to thepH value of the guiding fluid. The parameters of the graphicrepresentation are time, relative concentration change C/C_(o) andabsolute deviation (abs (pH-IP)) between the IP,and the pH value. It canbe seen that, even with slight deviations of the IP from the pH value,the concentration of the particular component fast reduces to zerobecause of the strong effect of the electric field.

In the continuous method the surrounding solution enriching itself withthe separated components is simultaneously replaced by fresh surroundingsolution.

FIG. 5 shows experimental confirmation of the high speed of separationin an example with a hollow fiber that is permeable to the components. Acontinuous sample stream is conducted through the hollow fiber anddetected at the downstream end without the influence of a field (point 1to 2). After application of the electric field (point 2) the protein isremoved entirely from the hollow fiber (reduction in absorption) with alow response time (of the order of a minute) because the pH value of thesurrounding solution does not correspond to the IP of the protein.

FIG. 6 is a schematic of a separation device according to the inventionfor separation of ampholytic molecules as a function of IP. The hollowfiber 61 and two electrodes 62 a, 62 b are arranged in an enclosure 64.The hollow fiber and the electrodes are parallel to one another, thehollow fiber being between the electrodes. The enclosure 64 is filledwith the surrounding solution 63 and joined to a reservoir 66. The pHsetting device 67 sets the pH value of the surrounding solution. Becauseof the semi-permeability of the hollow fiber, the pH value of theguiding fluid tracks the pH value of the surrounding solution. At leastone stirring means 65 is provided to ensure spatial pH constancy. At theupstream end of the hollow fiber 61 there is a pump 68 to convey theguiding fluid 69. The pump 68 is joined by a connection 610 (eg hose) tothe enclosure 64 and/or to a separate fluid reservoir 611. As analternative to the pump 68 for advancing the guiding fluid, a suctiondevice can be provided at the downstream end of the hollow fiber, thusensuring the necessary flow of the guiding fluid in the right direction.

Introduction of the sample 612 to the guiding fluid stream as a narrowband is through a three-way injector block 613 fitted with a means ofdosing (eg Hamilton injector 614). But microducts can also be providedin the hollow fiber wall for sample introduction.

At the downstream end of the hollow fiber there is a detector unit 615for identifying the substances in the lumen after passing through thehollow fiber, and a three-way cock 616 for controlled transfer of fluidfrom the hollow fiber into a fraction collector 617. The detector unit615 can be configured for opto-spectroscopic measurement for example. Ifthe hollow fiber consists of UV-permeable glass, proteins withabsorption in the UV band can be detected direct in the hollow fiber.

Particles with an IP differing from the pH value are fixed on the innerwall of the fiber. Particles with an IP corresponding to the pH valueare able to pass through the fiber unhindered. By variation of the pHvalue during separation it is possible to separate individual fractions,which then are rinsed one after the other from the fiber volume.

The device according to FIG. 6, depending on the form that the hollowfiber takes, can be used as both a separating device and a purifyingdevice for the methods explained above. It is possible to subdivide thereaction cavity so that it will hold several surrounding solutionsdiffering in their pH value. The electrodes are then interrupted anddriven separately as appropriate. Several devices as in FIG. 6 can alsobe cascaded.

In the design of the collection arrangement as a hollow fiber, accordingto the invention, the guiding fluid possesses a flow cross-section thatis much less than the length of the separation path. Thus theelectrodes, between which the hollow fiber is located, can be arrangedwith a small spacing so that small voltages suffice to generate highfield strength. Typical separator arrangements can be operated withvoltage between 1 and 10 V for example. Such small voltages avoidproduction of excessive heat and generation of electrolysis products,thus relaxing conditions for samples sensitive to heat. Compared to cIFFtechnology, which was referred to at the beginning, the inventionreduces the gap across which an electric field is applied by about threeorders of magnitude.

Further benefits of the invention are the short analysis or separationtimes (approx. 10 min or less), the possibility of automated andpermanent detection in a flow-through system, and the easy handling withconventional, elaborate gel or IPG-IEF techniques. Furthermore, theinvention allows separated components to be temporarily retained andreleased again after a predetermined field or pH alteration.

A further variant of isoelectric separation according to the inventionconsists in a concentrating procedure. A diluted solution with one ormore components flows continuously through the hollow fiber and isexposed to the influence of the field. The non-isoelectric componentsare fixed spatially on the hollow fiber wall. Following this flow thefield is removed and a solution (rinse) flows through the hollow fiberthat is lens than the total volume of the previous diluted solution. Therinse detaches the molecules fixed to the wall, which are thus releasedand leave the fiber, resulting in a solution of greater concentrationthan the original diluted solution.

Another application is in the acceleration of dialysis processes. If theionic concentration of the surrounding solution is less than that of theguiding fluid, ions will diffuse from the hollow fiber. This process canbe very much accelerated by the electric field.

With the device presented here it is also possible to characterize asubstance by its isoelectric parameters. This is of special interestwhen characterizing isoenzymes.

In what follows, modifications of the device according to FIG. 6 areexplained with reference to FIG. 7 through FIG. 12.

FIG. 7 shows an example of an implementation in which a bent hollowfiber 71 is arranged in the enclosure 72. The hollow fiber has a spiralshape. The electrodes 73 a, 73 b are matched to the geometry of thehollow fiber. In this example the electrodes 73 are ring electrodes,just covering the spiral cross-section. The spiral shape of the fibermakes it possible to separate larger amounts of sample simultaneously,because a larger amount of sample can be retained in the region of thewall where the flow velocity is zero (see FIG. 1). On a micropreparativescale this also allows continuous introduction of the sample.

FIG. 8 also shows a spirally shaped hollow fiber 81 wound onto acylindrically shaped electrode 82 a. The counter-electrode 82 b (showndotted) can be pushed over the arrangement as a hollow cylinder forexample. The surrounding solution can flow through the gap left betweenthe electrodes. The arrangement according to FIG. 8 is characterized byhigh charge or separation capacity plus a small space requirement andlow voltages.

The electrodes and collection means, described up to now as separatecomponents, can also be joined together as in FIG. 9. FIG. 9 shows, thecross-section of a hollow fiber 91 with electrodes 92 a, 92 b, which aredeposited as a thin layer on the outer wall of the hollow fiber. In thisway even smaller voltages can be used to produce high field strength.The surrounding solution influences the inside of the hollow fiberthrough the regions of the hollow fiber that have been left free.However, it is also possible to operate the system according to theinvention without a surrounding solution.

FIG. 10 shows the combination of a separating device (reaction cavity101 with hollow fiber and electrodes 102 a, 102 b) with a concentrationboosting device 104. This combined arrangement is operated so that, tobegin with, there is fractionating of a substance mix according to theinvention in the separation region. In the concentration boosting device104, whose surrounding solution 105 has a different pH value to thesurrounding solution 103 in the separation region, the purified fractioncan be fixed with the electrodes 106 a, 106 b as a narrow band and,after removal of the electric field on these electrodes, be applied tothe fraction collector 107 as a concentrated fraction.

The multi-compartment system according to FIG. 11 implements acounterflow principle where the surrounding solution 114 in theparticular reaction cavity is set into flow motion. The direction offlow should be opposite to the direction of flow in the hollow fiber112. A membrane 113 a, 113 b is placed between the electrodes and thehollow fiber 112 to intercept any electrolysis products appearing on theelectrodes 111 a, 111 b. The flow of the surrounding solution alsoserves to stabilize the pH in the reaction cavity.

In the invention it is not absolutely necessary for the guiding fluid toflow, it can also be static. It is also possible to move the collectionmeans relative to the guiding fluid. This is illustrated in FIG. 12. Achamber 123 is filled with a protein mix 121 and is demarcated bypermeable membranes 122. The chamber 123 is brought into thepH-regulated region 124. When the electric field is applied throughelectrodes 125 a, 125 b, single proteins drift, according to theprinciple of the invention, to the membranes 122. The membranes can beprovided with movable, protein-binding intermediary membranes that arewithdrawn in the direction of the arrow once they are occupied by theseparated proteins. Alternatively it is possible to provide an elutionstream in the direction of the arrow to rinse the separated proteins outof the membrane region.

The invention also foresees providing a separating device with several,cascaded collection means or several separation regions arranged inserial or parallel. Instead of the described hollow fiber, thecollection means can also be formed of any porous membranes or layersthat have appropriate compartments for the guiding fluid, next to whichthe electrodes are placed.

The semi-permeability of the collection means (eg of the hollow fiber)can vary over their length in the direction of flow so that section bysection certain ions of the guiding fluid and/or surrounding solutionand/or only certain molecules are allowed to pass.

It is also possible to modify the inner wall of the hollow fiber bychemical or physical methods to alter the effect of the electric field.The inside of the hollow fiber can be filled with gels, microparticlesuspensions or granulates, or a gel can be added to the reaction cavitytoo. The hollow fiber can be wound at a certain spacing round anelectrode of any shape and the counter-electrode can be matched to thisgeometry.

The collection means can be formed of a multi-fiber array consisting ofa large number of hollow fibers, through all of which the guiding fluidflows. The flow through the fibers is at different rates, however, sothat the efficiency of separation can be influenced as a function ofsubstance.

A DC voltage, an AC voltage or any other programmable voltage can beapplied to the (at least two) electrodes so that the field strength inthe guiding fluid is varied accordingly. This allows the formation ofpredetermined separation patterns on the collection means.

The electrodes can have modified surfaces or be of modified materials toreduce any electrolytic reactions that might appear. The electrodes canbe porous or of layers (eg gels, spacers).

The collection means can be bordered by flat membranes forming acompartment for the guiding fluid. The compartment can be subdividedwith the aid of further membranes to hold different protein fractions.In an arrangement of this kind the sample to be separated can beintroduced on a membrane that has absorbed the sample.

A special advantage of the separating device according to the inventionlies in miniaturization, in a preferred implementation it is intended toform and drive at least parts of the electrodes with electroniccomponents like transistors or diodes, and to produce thethree-dimensional arrangement of the electrodes, ducts, hollow fiberholders and reaction cavity using the technologies of semiconductorstructuring.

What is claimed is:
 1. A method for isoelectric particle separation,said method comprising the steps of: providing collection means arrangedadjacent to a fluid and adapted for soluble fixing of the particles inthe fluid, wherein said collection means comprise at least onesemipermeable or porous wall, along which the fluid with the particlesto be separated flows with a flow velocity, and wherein electrode meansextend, at least in part, along a direction of flow over the length ofthe semipermeable or porous wall, providing a fluid containing particlesthat have a net charge or charge density that is a function of the pHvalue of their surroundings, setting the pH value of the fluid,imparting motion to the particle-containing fluid, generating electricfield forces by said electrode means, permitting at least onepredetermined particle type to undergo a change in motion as a functionof charge through the effect of the electric field forces, permittingthe particle type undergoing the change in motion by the electric fieldforces to move to said collection means, retaining said particle type onthe collection means at least temporarily, and reducing or dropping tozero temporarily the flow velocity of the fluid when the particles to beseparated are exposed to the electric field forces in order to enhanceseparation of the particles being separated while the fluid with theparticles to be separated flows along the semipermeable or porous wall.2. The method according to claim 1, wherein the moved particle typecomprises particles whose isoelectric point does not correspond to thepH value of the fluid.
 3. The method according to claim 1, wherein theparticles to be separated comprise ampholytic molecules or otherparticles, synthetic particles or biological cells, viruses, or otherbiological objects whose electrical characteristics correspond to theelectrical characteristics of ampholytic molecules.